title: Transcription factors Sp1 and Hif2α mediate induction of the copper-transporting ATPase (Atp7a) gene in intestinal epithelial cells during hypoxia.
abstract: Genes with G/C-rich promoters were up-regulated in the duodenal epithelium of iron-deficient rats including those encoding iron (e.g. Dmt1 and Dcytb) and copper (e.g. Atp7a and Mt1) metabolism-related proteins. It was shown previously that an intestinal copper transporter (Atp7a) was co-regulated with iron transport-related genes by a hypoxia-inducible transcription factor, Hif2α. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iron deprivation/hypoxia. Initial studies in IEC-6 cells showed that mithramycin, an Sp1 inhibitor, reduced expression of Atp7a and iron transport-related genes (Dmt1, Dcytb, and Fpn1) and blocked their induction by CoCl2, a hypoxia mimetic. Consistent with this, overexpression of Sp1 increased endogenous Atp7a mRNA and protein expression and stimulated Atp7a, Dmt1, and Dcytb promoter activity. Site-directed mutagenesis and functional analysis of a basal Atp7a promoter construct revealed four functional Sp1 binding sites that were necessary for Hif2α-mediated induction of promoter activity. Furthermore, chromatin immunoprecipitation (ChIP) assays confirmed that Sp1 specifically interacts with the Atp7a promoter in IEC-6 cells and in rat duodenal enterocytes. This investigation has thus revealed a novel aspect of Atp7a gene regulation in which Sp1 may be necessary for the HIF-mediated induction of gene transcription during iron deficiency/hypoxia. Understanding regulation of Atp7a expression may help further clarify the physiological role of copper in the maintenance of iron homeostasis. Furthermore, this Sp1/Hif2α regulatory mechanism may have broader implications for understanding the genetic response of the intestinal epithelium to maintain whole-body iron homeostasis during states of deficiency.
authors.affiliation: Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Liwei
authors.initials: L
authors.lastname: Xie
authors.forename: James F
authors.initials: JF
authors.lastname: Collins
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D007413:Intestinal Mucosa / Q000166:cytology*; D007501:Iron / Q000378:metabolism; D000090463:Iron Deficiencies; D010766:Phosphorylation / Q000187:drug effects; D008926:Plicamycin / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D000251:Adenosine Triphosphatases; D051792:Basic Helix-Loop-Helix Proteins; D027682:Cation Transport Proteins; D003035:Cobalt; D003300:Copper; D000073840:Copper-Transporting ATPases; D007501:Iron; D008926:Plicamycin; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D000099162:Endothelial PAS Domain-Containing Protein 1; C486202:Atp7a protein, rat; C018021:cobaltous chloride
keywords: Chromatin Immunoprecipitation (ChIP); Cobalt Chloride; Hypoxia-inducible Factor (HIF); IEC-6 Cells; Intestine; Iron; Transcription Coactivators